Potent and reversible lentiviral vector restriction in murine induced pluripotent stem cells
© The Author(s) 2017
Received: 6 December 2016
Accepted: 23 May 2017
Published: 31 May 2017
The Correction to this article has been published in Retrovirology 2017 14:48
Retroviral vectors are derived from wild-type retroviruses, can be used to study retrovirus-host interactions and are effective tools in gene and cell therapy. However, numerous cell types are resistant or less permissive to retrovirus infection due to the presence of active defense mechanisms, or the absence of important cellular host co-factors. In contrast to multipotent stem cells, pluripotent stem cells (PSC) have potential to differentiate into all three germ layers. Much remains to be elucidated in the field of anti-viral immunity in stem cells, especially in PSC.
In this study, we report that transduction with HIV-1-based, lentiviral vectors (LV) is impaired in murine PSC. Analyses of early retroviral events in induced pluripotent stem cells (iPSC) revealed that the restriction is independent of envelope choice and does not affect reverse transcription, but perturbs nuclear entry and proviral integration. Proteasomal inhibition by MG132 could not circumvent the restriction. However, prevention of cyclophilin A (CypA) binding to the HIV-1 capsid via use of either a CypA inhibitor (cyclosporine A) or CypA-independent capsid mutants improved transduction. In addition, application of higher vector doses also increased transduction. Our data revealed a CypA mediated restriction in iPSC, which was acquired during reprogramming, associated with pluripotency and relieved upon subsequent differentiation.
We showed that murine PSC and iPSC are less susceptible to LV. The block observed in iPSC was CypA-dependent and resulted in reduced nuclear entry of viral DNA and proviral integration. Our study helps to improve transduction of murine pluripotent cells with HIV-1-based vectors and contributes to our understanding of retrovirus-host interactions in PSC.
In addition to their function as gene transfer vehicles, retroviral vectors can be utilized as tools to investigate specific vector-host interactions. Retroviral vectors are derived from their respective wild-type (wt) viruses by removal of nucleotide sequences in the RNA genomes, which are not essential for transgene delivery. Retroviral vectors are packaged into viral particles composed of structural and enzymatic proteins (Gag-Pol) and follow the natural course of the early retroviral life cycle. This includes cell entry, followed by a poorly-characterized uncoating process of the viral capsid, reverse transcription (RT) of the viral RNA genome into double-stranded DNA, nuclear entry of the viral DNA and stable, proviral integration into the host genome .
In this study, we investigated vector-host interactions in pluripotent stem cells (PSC). These cells naturally exist in early embryonic development and give rise to all three germ layers. PSC represent the equivalent to the inner cell mass of the blastocyst and possess major differentiation potential. Among PSC, induced pluripotent stem cells (iPSC) represent a prominent target cell type and cell source as they are easily generated by reprogramming of differentiated cells, a process mediated by ectopic expression of specific transcription factors . After successful reprogramming, iPSC obtain full pluripotency capacity and can differentiate into cells of all three germ layers in vitro as well as in vivo . Therefore, iPSC display a suitable alternative to embryonic stem cells (ESC) for the study of cellular differentiation and disease modeling. Additionally, iPSC are promising resources for cell and gene therapy applications, e.g. innovative cell transplants mediated by retroviral gene transfer.
Retroviral vector application and their gene transfer efficiency are closely dependent on virus-host cell interactions that either support or inhibit efficient infection. Retroviral replication is supported by host co-factors, which are crucial for successful infection. A prominent retroviral host co-factor is cyclophilin A (CypA), which directly binds to an exposed loop of the human immunodeficiency virus type 1 (HIV-1) capsid protein [4, 5]. CypA was described to support early events of HIV-1 infection, such as uncoating and RT initiation [6, 7]. In addition, it was shown that the interaction of CypA with other host co-factors determined the route of nuclear entry and led to alterations in integration site targeting . Furthermore, capsid-associated CypA can shield HIV-1 against cellular recognition and prevent a type 1 interferon response . In contrast, CypA-capsid binding can also inhibit HIV-1 infectivity in a species-specific manner by increasing the sensitivity to certain restriction factors [9–11]. Restriction factors are part of the cell autonomous immunity, are expressed by host cells and inhibit viral replication by inducing viral degradation, transcriptional silencing of integrated proviruses or initiation of anti-viral interferon responses. In this regard, previously identified restriction factors in PSC include the Trim28/ZFP809 complex [12, 13] and YY1 . Both factors target murine leukemia virus (MLV) in murine ESC. However, the role of cell autonomous immunity in PSC is only incompletely understood and so far not described for HIV-1.
Here, we describe an HIV-1-based vector restriction found in iPSC as well as in ESC. Focusing on iPSC, we identified a CypA-dependent restriction mechanism that impaired nuclear entry and reduced proviral integration. The restriction could be decreased by application of high vector doses, by prevention of CypA binding to the HIV-1 capsid or by iPSC differentiation.
Murine iPSC exhibit a potent block to HIV-1-based vectors
LV restriction is observed exclusively in the pluripotent state of a cell
LV transduction rate in murine iPSC is enhanced by high vector doses and CSA treatment
CypA-capsid interaction mediates LV restriction in iPSC
Low efficiency of LV transduction in iPSC is associated with reduced levels of 2-LTR circles and proviral DNA integration
The fact that efficiently reverse transcribed proviral DNA of LV failed to integrate into the chromatin of iPSC (Figs. 1c, 5d) suggests inefficient cytoplasmic trafficking, nuclear entry and/or integration. Our 2-LTR circle analyses at different time points after transduction (6, 12, 24, 48 h) revealed significantly reduced levels in untreated compared to CSA-treated cells (Fig. 5c). In addition, cells treated with the CSA/Raltegravir combination exhibited a pronounced increase of 2-LTR circles when compared to cells treated with Raltegravir application alone (see Additional file 4A). These findings clearly support that the mechanism responsible for the LV restriction in iPSC involved inefficient nuclear entry and that CSA relieved this hindrance. This could be explained by unproductive nuclear entry due to misdirected cytoplasmic trafficking to the nucleus, trapping of the PIC or impaired translocation into the nucleus. Alternatively, it is possible that, in addition to the nuclear entry block, there is an intranuclear block (e.g. caused by altered nuclear trafficking or perturbed integration), which is supported by different ratios of 2-LTR circles and proviral integration levels (see Additional file 4A and Fig. 5d). However, we hypothesize that the main block affects the nuclear entry step.
We identified the CypA-capsid interaction as a key event that mediated the restriction. Prevention of CypA-capsid interaction by CSA or specific CypA-independent capsid mutants led to a significant increase in transduction (Figs. 3e, 4b). Our data provide evidence that this interaction is most crucial relatively early after cell entry, but we also observed supportive effects of CSA at the latest time point measured (12 h) (Fig. 4a). Interestingly, CypA expression levels were comparable in restricted iPSC and permissive cells, thus there was no correlation between CypA levels and the block (Fig. 4c). Furthermore, we were unable to saturate the block by pretransduction with high doses (MOI 1000) of LV in our abrogation experiments (Fig. 3c). Potential explanations for the lack of an abrogation phenotype include: (I) even higher concentrations are needed to saturate the potential factor; (II) the block is not saturable; (III) using our doses, other essential cellular pathways and receptors are blocked and are not recycled in a timely manner in the 6 h time window before the second transduction.
The restriction was relieved by use of CypA-independent capsid mutants (P90A, G89V, H2.1, A88T) (Fig. 4b). In contrast, the N74D capsid mutant, which binds CypA to a lower extent than the wt capsid, revealed no beneficial effect on transduction . This suggests that the amount of capsid-bound CypA is important for the restriction. Furthermore, it was previously reported that the capsid mutants P90A and G89V utilize different nuclear entry routes compared to wt capsid . We can therefore envision that, in addition to avoiding binding of CypA molecules to the capsid, also the different nuclear entry routes could play a role in relieving the restrictive phenotype of LV in iPSC.
In addition to a potential restriction factor, the block may also be explained by the lack of a necessary host co-factor. Thus, we performed a detailed microarray analysis of different iPSC clones and their parental fibroblasts and compared expression of previously reported HIV-1 host co-factors for nuclear entry and integration as well as of a set of murine nucleoporins (see Additional file 5). When compared to adult fibroblasts, iPSC exhibited similar or even higher expression levels of examined nucleoporins and important host co-factors for nuclear entry and integration. This suggests that differences in expression levels of the analyzed HIV-1 host co-factors and nucleoporins do not contribute to the underlying mechanism of the observed block.
As previously described, somatic and pluripotent stem cells evolved anti-viral defense mechanisms [12, 14, 31, 46–48]. The novel LV block we identified in iPSC is acquired during the process of reprogramming and relieved upon differentiation (Fig. 2a–c). In our experiments ESC and iPSC exhibited similarly low LV transduction rates and integration events compared to GV (Fig. 1c, e). In contrast, this phenomenon is less pronounced in ECC (Fig. 2d) . This might be explained by the fact that ECC are supposed to represent a more differentiated status compared to PSC, including iPSC and ESC. Thus, we hypothesize that the LV restriction in iPSC may be associated with the pluripotent state.
Although, murine cells are not the natural host for HIV-1, lentiviruses were isolated from several species other than primates and the CypA-capsid interaction was also discovered to be highly conserved . Moreover, the presence of endogenous lentiviruses in the genomes of a variety of different species demonstrates that lentiviruses are more widespread than previously assumed. Therefore, an active lentiviral defense mechanism in murine PSC is imaginable. Likewise, much remains to be discovered regarding anti-viral defense mechanisms in stem cells, especially in PSC. This knowledge can help to develop more efficacious anti-viral treatments in order to modulate cell autonomous immunity, and also increase gene transfer efficiencies for studies in basic biology and therapeutic approaches. Our findings document the characteristics of the restriction against HIV-1 found in PSC and contribute to our understanding of HIV-1-host interactions.
We identified CypA as a key player in LV restriction in iPSC and discovered impaired nuclear entry and proviral integration as underlying mechanisms for low transduction rates. To the best of our knowledge an HIV-1 block in PSC has not been previously described. Furthermore, the restriction was not observed in parental fibroblasts or differentiated iPSC progeny, which suggests that LV restriction is acquired through reprogramming into the pluripotent state. While our investigation demonstrated a novel HIV-1 restriction in murine PSC, it remained unclear whether the restriction is caused by a restriction factor or the absence of an essential host co-factor. While CypA was previously described to either support or inhibit wt HIV-1 infection in a cell type-dependent manner, the CypA-capsid interaction, we identified to be responsible for the restriction in iPSC, differs from previously described studies and clearly contributes to our understanding of the anti-viral activity in PSC.
Retroviral vector plasmids
Lentiviral vector plasmids pRRL.PPT.EFS.EGFP.pre [50–52] and pRRL.PPT.SFFV.DsRedExpress.pre  were referred to as LV and LV.DsRed, respectively. Gammaretroviral vectors pSERS11.EFS.EGFP.pre  and pSF91.DsRedExpress  were termed GV and GV.DsRed. Except for the LTR-driven GV.DsRed, all vectors had SIN designs with deletions of the viral U3 regions (∆U3), instead containing the internal EFS promoter (short version/250 bp fragment of promoter/enhancer sequences from the human elongation factor 1 alpha gene) or, for LV.DsRed, the SFFV promoter (promoter/enhancer sequences from spleen focus forming virus). For reprogramming, the lentiviral “4-in-1” construct pRRL.PPT.SF.hOKSMco.id.Tom.pre.F+F3, co-expressing codon-optimized versions of the transcription factors Oct4, Klf4, Sox2 and wt c-Myc, was used [15, 54]. The construct contained heterospecific FRT sites (F and F3) to allow Flp recombinase-mediated excision of the reprogramming cassette [15, 55].
Cells and cultivation
Human 293T (embryonic kidney cell line) , human HT1080eCat (fibroblast cell line HT1080  expressing the murine ecotropic receptor mCAT-1 ) and the murine ECC line F9 (kindly provided by S. P. Goff, Columbia University Medical Center, New York, NY, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Biochrom GmbH, Berlin, Germany) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (GE Healthcare Europe GmbH, Freiburg, Germany), 100 U/mL penicillin, 100 µg/mL streptomycin and 1 mM sodium pyruvate (all from PAN-Biotech, Aidenbach, Germany). CF-1 Mefs (MTI Global Stem, Gaithersburg, MD, USA) and C3H Mefs (kindly provided by T. Cantz, Hannover Medical School, Hannover, Germany) as well as freshly isolated murine adult fibroblasts (C57BL/6 p14f/f) were grown in low glucose DMEM (PAN-Biotech) supplemented with 15% heat-inactivated FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, 2 mM l-glutamine, 1% MEM non-essential amino acids solution (Gibco Life Technologies GmbH, Darmstadt, Germany) and 100 µM beta-mercaptoethanol (Sigma-Aldrich, Munich, Germany) on gelatin pre-coated (0.1% gelatin in phosphate buffered saline (PBS)) wells or flasks. PSC were co-cultured with irradiated (30 Gy) C3H Mefs in ESC medium composed of Knockout DMEM (Gibco Life Technologies) with 15% ESC-tested and heat-inactivated FBS (GE Healthcare Europe GmbH), 2 mM l-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, 1% MEM non-essential amino acids, 100 µM beta-mercaptoethanol and 103 U/mL LIF (kindly provided by the Department of Technical Chemistry, Leibniz University Hannover, Hannover, Germany). The following murine PSC were used in this study: iPSC clone #1 (C57BL/6 mouse strain), clones #2, #2EX (excised reprogramming cassette) and #3 (C57BL/6 p14f/f, wt version without conditional knockout of the p14 gene, reprogrammed from adult fibroblasts, mice kindly provided by D. Kotlarz and C. Klein, Ludwig Maximilian University, Munich, Germany), clone #4 (reprogrammed from C3H Mefs) and ESC (C57BL/6 mouse strain, kindly provided by I. Prinz, Hannover Medical School).
Retroviral particle production
One day before transfection, 5 × 106 293T cells were seeded per 10-cm dish. Transfection was performed based on the calcium phosphate precipitation method assisted by 25 µM chloroquine (Sigma-Aldrich). For LV or LV.DsRed, packaging cells were co-transfected with 5 μg LV plasmid, 5 μg pRSV-Rev (kindly provided by T. Hope, Northwestern University, Chicago, IL, USA), 1.5 μg pMD.G (VSVg) , 12 μg wt pcDNA3.gp.4xCTE (HIV-1 Gag-Pol)  or CypA-independent capsid mutant Gag-Pol P90A, G89V, A88T or H2.1 or the N74D mutant [36, 38, 39, 41]. Mutations were introduced by overlap extension PCR and confirmed by sequencing. For production of LV-Ampho(tropic) and LV-Eco(tropic) particles, 2 μg ecotropic  or 2 μg amphotropic  MLV envelope encoding plasmids were used. To package VSVg pseudotyped GV or GV.DsRed particles, 5 μg GV plasmid, 7 μg pcDNA3.MLV.GP (MLV Gag-Pol)  and 1.5 μg pMD.G were co-transfected. Supernatants were harvested 36 and 48 h after transfection, filtered through 0.22 μm pore-size-filters and concentrated (100×) by ultracentrifugation (2 h, 82,740×g, 4 °C) (SW32Ti rotor; Beckman Coulter GmbH, Krefeld, Germany). Viral pellets were resuspended in ESC medium and stored in aliquots at −80 °C until further usage.
Retroviral particle titration and transduction
One day before titration of vector supernatants, 7 × 104 HT1080eCat cells were seeded per well of a 12-well plate. The next day, the culture medium was removed and serial dilutions of vector supernatants were added to the cells. Viral transduction of cells was performed in the presence of 4 µg/mL protamine sulfate and spin-inoculation (1 h, 863×g, 37 °C). Three days after transduction, the percentage of fluorescent cells was analyzed by flow cytometry. Vector particle titers were calculated only including samples with < 30% transduced cells to avoid multiple proviral integration events per cell and false titer estimation. Mefs (3 × 104 cells per well of a 12-well plate) or ECC (5 × 104 cells per well of a 12-well plate) were seeded the day before transduction. Specific amounts of vector particles per cell, referred to as MOI, were applied and cells were treated as described above for titration. PSC were transduced in single cell suspension and separated with 0.5% Trypsin-EDTA in PBS (Gibco Life Technologies GmbH) on the day of transduction. Afterwards, PSC were centrifuged (138×g, 5 min) and depleted from feeder cells. Depletion was performed by incubating the cells on a 15-cm plate (40 min, 37 °C, 5% CO2) to separate the faster attaching feeder from PSC. After applying vector supernatants at specific MOI to 3 × 104 cells (12-well plate) and adding 4 µg/mL protamine sulfate, a spin-inoculation step (1 h, 863×g, 37 °C) and an incubation step (1 h, 37 °C, 5% CO2) were performed, before PSC were added to feeder cells. The next day, PSC had attached to the feeder cell layer, and the virus-containing medium was replaced by fresh ESC medium. For small molecule supplemented transductions, cells were incubated 30 min prior to and during transduction with indicated concentrations of CSA (Sigma - Aldrich), MG132 (Calbiochem/Merck Millipore, Darmstadt, Germany), Nevirapine (Sigma-Aldrich) or Raltegravir (Santa Cruz Biotechnology, Dallas, TX, USA). Depending on the cell type and experiment, cells were processed by flow cytometry 2 days (differentiated cultures), 3 days (HT1080eCat titration, C3H Mefs), 5–8 days (adult fibroblasts, ESC, ECC, iPSC) or at indicated time points after transduction.
Reprogramming murine primary fibroblasts to iPSC
Differentiation of iPSC
To induce differentiation, iPSC were depleted from feeder cells, seeded in gelatin pre-coated (0.1% gelatin in PBS) 12-well plates (5 × 104 per well) and cultured in ESC medium without LIF and feeder cells. Medium was exchanged daily and cells were split in the range of 1:5–1:20 on days 3–4 and 5–6. Prior to transduction of differentiated cultures, cells were stained for SSEA1 expression and analyzed by flow cytometry to investigate their stem cell status. Differentiated cultures were transduced with retroviral particles (MOI 100) encoding EGFP or DsRedExpress 6–9 days after starting differentiation. Cells transduced with DsRedExpress encoding vectors were additionally stained with Stain Alive SSEA1 Antibody DyLight 488 according to the manufacturer’s protocol (Stemgent, Cambridge, MA, USA) and analyzed by fluorescence microscopy 2 days after transduction. Pictures were acquired with Axio Observer Z1 (Carl Zeiss AG, Jena, Germany) using Zeiss filter sets 43 (DsRedExpress) and 38 (DyLight 488) and AxioVision 4.8 software. Cells transduced with EGFP encoding vectors were analyzed by flow cytometry 2 days after transduction.
Cells were harvested and subsequently analyzed by flow cytometry (FACS Calibur, Becton-Dickinson, Heidelberg, Germany) using FlowJo software (Tree Star Inc, Ashland, OR, USA). In addition, PSC or differentiated cultures were stained with an Alexa 647 labeled SSEA1 antibody (1.5 ng per sample, 30 min, 4 °C) (eBioscience, San Diego, CA, USA). Samples were pre-gated for viable cells. Data from total viable cell populations with at least 50% SSEA1 positive cells were included in the analyses. The range of SSEA1 positive cells constituted mostly 70–100% throughout the study. The 70–100% range derives from differences between independent transduction experiments, but there were equivalent amounts of SSEA1 positive cells within each experiment. Cells, double positive for SSEA1 and EGFP, are shown in the graphs.
SYBR Green-based quantitative real-time PCR for detection of mean vector copy number per cell
iPSC were transduced with LV and GV and DNA was isolated 6–8 days after transduction. Vector copies per cell shown in Fig. 1c, e and Additional file 4B were determined by SYBR Green-based quantitative real-time PCR based on EGFP copies (Applied Biosystems, Darmstadt, Germany) using the QuantiTect SYBR-Green PCR Kit (Qiagen, Hilden, Germany) normalized to endogenous PTBP2 (EGFP for: 5′CTATATCATGGCCGACAAGCAGA3′, rev: 5′GGACTGGGTGCTCAGGTAGTGG3′; PTBP2 for: 5′GTCTCCATTCCCTATGTTCATGC3′, rev: 5′GTTCCCGCAGAATGGTGAGGTG3′). Master Mix preparation and PCR were performed as instructed by the manufacturer. Quantification of the mean vector copy number per cell were determined based on the comparative 2−∆∆Ct method and based on a reference plasmid containing EGFP and PTBP2 sequences [62–64].
Analysis of RT products, 2-LTR circles and proviral integration by TaqMan-based quantitative real-time PCR
Vector supernatants were applied (MOI 10) to iPSC and CF-1 Mefs and remaining vector particles were removed by washing the cells twice with PBS 6 h after transduction. Cells were harvested at indicated time points. To test for plasmid contamination of vector supernatants, equivalent volumes of vector supernatants were heat-inactivated at 65 °C for 1 h and included in the analyses (plasmid ctrl). DNA isolation was performed with the QIAamp DNA Blood Mini Kit (Qiagen) according to the manufacturer’s instructions. For TaqMan-based quantitative real-time PCR (Applied Biosystems), RT products were analyzed for early RT products (strong-stop DNA, for: 5′GCCTCAATAAAGCTTGCCTTGA3′, rev: 5′TGACTAAAAGGGTCTGAGGGATCT3′, probe: 5′AGAGTCACACAACAGACGGGCACACACTA3′), late RT products (U5/downstream PBS, for: 5′TAGTGTGTGCCCGTCTGTTG3′, rev: 5′GAGTCCTGCGTCGAGAGAG3′, probe: 5′TCCCTCAGACCCTTTTAGTCA3′) and 2-LTR circles (junction, for: 5′TAGTGTGTGCCCGTCTGTTG3′, rev: 5′CAGAGAGACCCAGTACAAGC3′, probe: 5′CTCTAGCAGTAACTGGAAGGGCT3′). PTBP2 served as a housekeeping control (for: 5′TCTCCATTCCCTATGTTCATGC3′, rev: 5′GTTCCCGCAGAATGGTGAGGTG3′, probe: 5′ATGTTCCTCGGACCAACTTG3′). RT products were relatively quantified using the comparative 2−∆∆Ct method [62, 63]. PCR was performed at 50 °C for 2 min and at 95 °C for 20 s followed by 60 cycles of 5 s at 95 °C, 20 s at 56 °C and 20 s at 65 °C.
B1-LTR PCR for analysis of proviral integration
Briefly, a first stage PCR was run with a LTR forward primer, which was designed to harbor a lambda-phage heel sequence at the 5′ end and LTR sequences at the 3′ end. In addition, two outward facing primers binding the highly redundant consensus sequence within the mouse B1 repetitive element were used in the first stage PCR. This was followed by a second stage TaqMan-based quantitative real-time PCR, which amplified the LTR using a lambda-specific forward and an LTR-specific reverse primer, scored using an LTR-specific probe. Primer sequences and protocol were used from Tervo et al. . For data analyses, genomic DNA from a SC-1 standard cell line, harboring three HIV-1-based vector integrations , was diluted over a range of concentrations covering three logs. Slope and y-intercept of the standard curve were used to determine proviral copies. PTBP2 served as a housekeeping control. To exclude plasmid contamination of vector supernatants, 5 µM Nevirapine was used as an RT inhibitor, for every time point with equivalent volumes of vector supernatants, and signals were subtracted from the total signal. For each sample, PCR controls, omitting the B1 forward and reverse primers or the Lambda-LTR forward primer, were performed in parallel during the first PCR stage. The approach omitting the Lambda-LTR forward primer did not reveal any signal. The signal achieved by omission of B1 forward and reverse primers was subtracted from the total signal for each sample.
Western blot analysis
Cells were harvested, washed with PBS and lysed by using radioimmunoprecipitation assay buffer supplemented with proteinase inhibitors (Complete Mini, Roche, Mannheim, Germany). Samples (15 µg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12.5%) and blotted onto nitrocellulose membranes (GE Healthcare Europe GmbH). Antibody probing was conducted with rabbit monoclonal anti-CypA (Cell Signaling Technology, Danvers, MA, USA) and rabbit polyclonal Erk2 (Santa Cruz Biotechnology) according to the manufacturer’s instructions. Goat anti-rabbit IgG conjugated with horseradish peroxidase was used as a secondary antibody. Quantitative detection was carried out using the Fusion Fx system (Peqlab GmbH/VWR Life Science Competence Center, Erlangen, Germany).
iPSC were pre-transduced with LV.DsRed or GV.DsRed at an MOI of 1000. After spin-inoculation cells were incubated at 37 °C and 5% CO2. After 6 h incubation time, iPSC were washed twice with PBS and a second transduction with LV was performed. LV encoding EGFP at an MOI of 100 were used for the second transduction. Flow cytometry analyses were conducted 4 days after transduction experiments.
Data were expressed as mean ± standard deviation. Where appropriate, we used one-way ANOVA with Tukey-Kramer post hoc test to adjust for multiplicity effects. When comparing all groups to one control group, we utilized one-way ANOVA with Dunnett post hoc test. For time course analyses, we utilized repeated measures one-way ANOVA with appropriate post hoc test. The unpaired t test was performed for comparison of two groups. In case of significantly different variances between the groups, the unpaired t test with Welch’s correction was applied. p values of ≤ 0.05 were considered significant (*), ≤ 0.01 very significant (**), ≤ 0.001 extremely significant (***), and ns was considered not significant. Supplementary material and methods are described in Additional file 6.
FG, MG, DH, SPG, A. Schambach designed the experiments and analyzed the data. FG performed the experiments Figs. 1, 2, 3, 4 and 5 and Additional files 1, 2, 3 and 4. TM cloned and validated the H2.1 capsid mutant. JWS illustrated Fig. 6 and helped to perform additional experiments for paper revision. JK generated and characterized iPSC clones #1, #2, #2EX and #3. DZ initially observed the LV block in iPSC. A. Schwarzer analyzed microarray data, performed bioinformatics and prepared Additional file 5. CG provided B1-LTR PCR protocol and contributed her expertise analyzing the data. FG, MG, DH, TM, JWS, SPG, and A. Schambach wrote the manuscript. All authors read and approved the final manuscript.
We thank Michael Morgan, Hildegard Büning and Verena Labenski for helpful discussion and assistance with the manuscript. We furthermore thank Thomas Neumann and Girmay Asgedom for technical assistance, Michael Rothe for consultation on quantitative PCR analysis and Steven Talbot for support and approval of statistical analyses. We are grateful to Eva Warlich for providing the C3H iPSC clone #4, to Daniel Griffin (Columbia Medical Center, New York, NY, USA) for the early RT TaqMan primer/probe, to Daniel Kotlarz and Christoph Klein (both Ludwig Maximilian University, Munich, Germany) for providing the C57BL/6 p14f/f mice as well as to Hanna-Mari Baldauf (Ludwig Maximilian University) for providing reference cell lines for the B1-LTR PCR. We would like to acknowledge the assistance of the Cell Sorting Core Facility of the Hannover Medical School supported in part by Braukmann-Wittenberg-Herz-Stiftung and Deutsche Forschungsgemeinschaft.
The authors declare that they have no competing interests.
Ethics approval and consent to participate
Isolation of murine adult fibroblasts was conducted in accordance with German law for animal protection and with the European Directive (2010/63/EU). All animal experiments were approved and permitted by the Lower Saxony State Office for Consumer Protection and Food Safety (LAVES).
This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB738, projects C4 and C9), Cluster of Excellence REBIRTH (EXC 62/1 SPP1230), the Bundesministerium für Bildung und Forschung (BMBF, IFB-Tx, PidNet), the DAAD (Modern Applications in Biotechnology, Short-Term USA Fellowship 2014), the European Union (FP7 project PERSIST) and NIH NCI (R01 CA30488). SPG is an investigator of the Howard Hughes Medical Institute. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
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